Staining is an important auxiliary technique used in identification, detection, visualization, labeling, targeting, and purification of biological samples. In addition, it is also widely used in microscopy to enhance contrast in the microscopic image. In particular Stains and dyes are frequently used in chemistry, biology and medicine to visualize and quantify nucleic acids, proteins and also other biological materials to highlight structures in the tissues for various reasons such as examining tissues, classifying cell populations, or organelles within individual cells. Stains are chemical compounds that exhibit a detectable response when contacted with a particular target. In the absence of the target, a stain does not exhibit the detectable response. These properties make stains valuable in the detection of the presence or absence of a particular target in a sample. The detectable response can be qualitative or quantitative, depending on the compound, target, and assay parameters.
The detection and quantitation of nucleic acids particularly DNA is a very common task in biotechnological research. Early chemical stains are effective at staining DNA, but also stain RNA. DNA and RNA are often obtained together when isolated from natural sources. Stains that are not selective for DNA make quantitation of the isolated DNA difficult, requiring a purification step to be performed prior to quantitation. Specialized research requires differentiation of different types of nucleic acids like single standard DNA, double standard DNA and plasmid DNA. The nucleic acid screening applications uses presently available toxic and mutagenic fluorescent stains by compromising on safety. The process involves cumbersome safety protocols and expensive disposal practices. In particular, fluorescent stains/dyes are widely used in nucleic acid research to visualize and quantify DNA/RNA fragments in agarose gels. Ethidium bromide (EtBr) has been the predominant dye used for nucleic acid screening for decades because of its low cost and sufficient sensitivity. However, the safety hazard and costs associated with decontamination and waste disposal can ultimately make the dye expensive and unsafe to researchers and environment.
Several alternate stains like SYBR group of stains, Gel red, Gel green, Pico green, Dimeric cyanines etc., have been developed to replace the highly toxic EtBr. Invariably, all these dyes are synthetic-aromatic ring structures highly sensitive to detect nucleic acids. Despite their higher sensitivity they express certain level of toxicity and mutagenicity. The safety claims projected by these dyes are in comparison with the highly toxic EtBr. Thus, there is no safe fluorescent stain available for safe screening of biological samples or nucleic acids.
The compound brazilein is derived from the bark of Caesalpinia sappan, a shrub found widely in Asia. Furthermore, the extraction of brazilein through a simple process from Caesalpinia sappan species and obtaining a good yield is quite cumbersome and difficult. There are reports on the extraction and separation of brazilin and brazilein compounds from Caesalpenia species (WO 2007066926 A1, WO/2007/066926). All these methods explain about the extraction and characterization of brazilein as a food and fabric staining agent. However, the nucleic acid staining property of brazilein in gel based systems and related applications was not attempted. Further, many different methods and strategies have been used in extracting and purifying brazilein compound from the Caesalpenia species. In CN102241660 (A), titled “Method for purifying Brazilein”, the referred invention explains about an extraction method for brazilein from brazil wood. The invention uses a combination of solvents including ethanol, ethyl ester to extract brazilein. The column purification step followed for the extraction protocol also involves acetonitrile, phosphoric acid as mobile phase and silica gel as filtering medium for eluting brazilein. The method also used preparative HPLC for purifying brazilein from the plant extract. This method is expensive and involves two purifying protocols leading to long and cumbersome procedure.
The invention referred in the patent no CN102219771A titled “Method for extracting brazilein from sappan wood”, claims a method for extracting brazilein from sappan wood. The method involves methanol as a primary solvent for treating raw material followed by macroporous resin based absorption for separating brazilein. Though the method used methanol as the solvent for purifying brazilein, the concentration, solvent combination and incubation duration employed is different, further the method uses several other steps for extraction including, addition of water for dispersion, use of macroporous resin for adsorption, gradient elution using methanol, crystalizing the crystals washing and dissolving the crystals and finally recrystallizing in ethanol and ethyl estate.
In WO 2007066926 A1 titled “a mass production method of brazilein from Caesalpinia sappan”, the invention claims an alcohol based brazilein extraction method for large scale production. However, the extraction involves two stage crystallization in which alcohol is the only solvent to purify brazilein.
In Korean patent 1020050120665, titled “Rapid and inexpensive mass production method of brazilein having high purity from Caesalpinia sappan” discloses a method of extracting brazilein using water or alcohol, however it requires recrystallization and use of controlled conditions and longer incubation periods.